POLYCYSTIC KIDNEY DISEASE 1 GENE AND USES THEREOF

Abstract: Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder which frequently results in renal failure, due to progressive cyst development. The major locus, PKD1, maps to 16p13.3. A chromosome translocation is identified associated with ADPKD which disrupts a gene (PBP), encoding a 14 kb transcript, in the PKD1 candidate region. Further mutations of the PBP gene were found in PKD1 patients confirming that PBP is the PKD1 gene. This gene is located adjacent to the tuberous sclerosis (2) locus in a genomic region that is reiterated more proximally on 16p. The duplicate area encodes three transcripts substantially homologous to the PKD1 transcript. Partial sequence analysis of the PKD1 transcript shows that it encodes a novel protein. Screening of actual or suspected ADPKD patients for normal or mutated PKD1 can be used for diagnostic purposes. PKD1-associated disorders such as ADPKD may be treated or prevented by PKD1 gene therapy and/or administration of functional PKD1 protein to affected cells.

Description: POLYCYSTIC KIDNEY DISEASE 1 GENE AND USES THEREOF The present invention relates to the polycystic kidney disease 1 (PKDl) gene, mutations thereof in patients having PKDl-associated disorders, the protein encoded by the PKDl gene, and their uses in diagnosis and therapy. Background to the Invention All references mentioned herebelow are listed in full at the end of the description which are herein incorporated by reference in their entirety. Except where the context clearly indicates otherwise, references to the PBP gene, transcript, sequence, protein or the like can be read as referring to the PKDl gene, transcript, sequence, protein or the like, respectively. A landmark study by Dalgaard, 1957 showed that autosomal dominant polycystic kidney disease (ADPKD) also termed adult polycystic kidney disease (APKD) is one of the commonest genetic diseases of man (approximately 1/1000 individuals affected). The major feature of this dominant disease is the development of cystic kidneys which commonly leads to renal failure in adult life. This simple description, however, belies the diverse systemic disorder, affecting many other organs (reviewed in Gabow, 1990) and one which occasionally presents in childhood (Fink, et al., 1993; Zerres, et al., 1993). Extrarenal manifestations include liver cysts (Milutinovic, et al . , 1980), and more rarely cysts of the pancreas (Gabow, 1993) and other organs. Intracranial aneurysms occur in approximately 5% of patients and are a significant cause of morbidity and mortality due to subarachnoid haemorrhage (Chapman, et al., 1992). More recently, an increased prevalence of cardiac valve defects (Hossack, et al., 1988), herniae (Gabow, 1990) and colonic diverticulae (Scheff, et al . , 1980) has been reported. The major cause of morbidity in ADPKD, however, is progressive renal disease characterised by the formation and enlargement of fluid filled cysts, resulting in grossly enlarged kidneys. Renal function deteriorates as normal tissue is compromised by cystic growth, resulting in end stage renal disease (ESRD) in more than 50% of patients by the age of 60 years (Gabow, et al . , 1992): ADPKD accounts for 8-10% of all renal transplantation and dialysis patients in Europe and the USA (Gabow, 1993). Biochemical studies have suggested several potential causes of cyst formation and development, including: abnormal epithelial cell growth, alterations to the extracellular matrix and changes in cellular polarity and secretion (reviewed in Gabow, 1991; Wilson and Sherwood, 1991). The primary defect in ADPKD, however, remains unclear and considerable effort has therefore been applied to identifying the defective gene(s) in this disorder by genetic approaches. The first step towards positional cloning of an ADPKD gene was the demonstration of linkage of one locus now designated the polycystic kidney disease 1 (PKDl) locus to the a globin cluster on the short arm of chromosome 16 (Reeders, et al., 1985). Subsequently, families with ADPKD unlinked to markers of 16p were described (Ki berling, et al., 1988; Romeo, et al . , 1988) and a second ADPKD locus (PKD2) has recently been assigned to chromosome region 4ql3- q23 (Kimberling, et al., 1993; Peters, et al., 1993). It is estimated that approximately 85% of ADPKD is due to PKDl (Peters and Sandkuijl, 1992) with PKD2 accounting for most of the remainder. PKD2 appears to be a milder condition with a later age of onset and ESRD (Parfrey,-et al., 1990; Gabow, et al., 1992; Ravine, et al., 1992). The position of the PKDl locus was refined to chromosome band 16pl3.3 and many markers were isolated from that region (Breuning, et al., 1987; Reeders , et al., 1988; Breuning, et al . , 1990; Germino, et al., 1990; Hyland, et al . , 1990; Himmelbauer, et al., 1991) . Their order, and the position of the PKDl locus, has been determined by extensive linkage analysis in normal and PKDl families and by the use of a panel of somatic cell hybrids (Reeders, et al., 1988; Breuning, et al., 1990; Germino, et al., 1990). An accurate long range restriction map (Harris, et al., 1990; Germino, et al. , 1992) has located the PKDl locus in an interval of approximately 600 kb between the markers GGG1 and SM7 (Harris, et al. , 1991; Somlo, et al . , 1992) (see Figure la). The density of CpG islands and identification of many RNA transcripts indicated that this area is rich in gene sequences. Germino et al (1992) estimated that the candidate region contains approximately 20 genes. Identification of the PKDl gene from within this area has thus proved difficult and other means to pinpoint the disease gene were sought. Linkage disequilibrium has been demonstrated between PKDl and the proximal marker VK5 , in a Scottish population (Pound, et al., 1992) and between PKDl and BLu24 (see Figure la), in a Spanish population (Peral, et al., 1994) . Studies with additional markers have shown evidence of a common ancestor in a proportion of each population (Peral, et al. , 1994; Snarey, et al. , 1994), but the association has not precisely positioned the PKDl locus. Disease associated genomic rearrangements, detected by cytogenetics or pulsed field gel electrophoresis (PFGE) have been instrumental in the identification of various genes associated with various genetic disorders. Hitherto, no such abnormal iti.es related to PKDl have been described. This situation contrasts with that for the tuberous sclerosis locus, which lies within 16pl3.3 (TSC2). In that case, TSC associated deletions were detected by PFGE within the interval thought to contain the PKDl gene and their characterisation was a significant step toward the rapid identification of the TSC2 gene (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). The TSC2 gene therefore maps within the candidate region for the hitherto unidentified PKDl gene; as polycystic kidneys are a feature common to TSC and ADPKD1 (Bernstein and Robbins, 1991) the possibility of an aetiological link, as proposed by Kandt et al. (1992), was considered. We have now identified a pedigree in which the two distinct phenotypes, typical ADPKD or TSC, are seen in different members. In this family, the two individuals with ADPKD are carriers of a balanced chromosome translocation with a breakpoint within 16pl3.3. We have located the chromosome 16 translocation breakpoint and a gene disrupted by this rearrangement has been defined; the discovery of additional mutations of that gene in other PKDl patients shows that we have identified the PKDl gene. Summary of the Invention Accordingly, in one aspect, this invention provides an isolated, purified or recombinant nucleic acid sequence comprising: - (a) a PKDl gene or its complementary strand, (b) a sequence substantially homologous to, or capable of hybridising to, a substantial portion of a molecule defined in (a) above, (c) a fragment of a molecule defined in (a) or (b) above. In particular, there is provided a sequence wherein the PKDl gene has the partial nucleic acid sequence according to Figure 7 and/or 10. Thp invention therefore includes a DNA molecule selected from: (a) a PKDl gene or its complementary strand, (b) a sequence substantially homologous to, or capable of hybridising to, a substantial portion of a molecule defined in (a) above, (c) a molecule coding for a polypeptide having the partial sequence of Figure 7, (d) genomic DNA corresponding to a molecule in (a) above; and (e) a fragment of a molecule defined in any of (a), (b), (c) or (d) above. The PKDl gene described herein is a gene found on human chromosone 16, and the results of familial studies described herein form the basis for concluding that this PKDl gene encodes a protein called PKDl protein which has a role in the prevention or suppression of ADPKD. The PKDl gene therefore includes the DNA sequences shown in Figures 7 and 10, and all functional equivalents. The gene furthermore includes regulatory regions which control the expression of the PKDl coding sequence, including promotor, enhancer and terminator regions. Other DNA sequences such as introns spliced from the end-product PKDl RNA transcript are also encompassed. Although work has been carried out in relation to the human gene, the corresponding genetic and functional sequences present in lower animals are also encompassed. The present invention therefore further provides a PKDl gene or its complementary strand having the partial sequence according to Figure 7. In particular, it provides a PKDl gene or its complementary strand having the partial sequence of Figures 7 and/or 10 which gene or strand is mutated in some ADPKD patients (more specifically, PKDl patients). The invention further provides a nucleic acid -sequence comprising a mutant PKDl gene, especially one selected from a -sequence c-omprising a partial sequence according to Figures 7 and/or 10 when: (a) [0X114] base pairs 1746-2192 as defined in Figure 7 are deleted (446bp); (b) [0X32] base pairs 3696-3831 as defined in Figure 7 are deleted by a splicing defect; (c) [0X875] about 5.5kb flanked by the two Xbal sites shown in Figure 3a are deleted and the EcoRl site separating the CWIO (41kb) and JH1 (18kb) sites is thereby absent (d) [WS53] about lOOkb extending between the JH1 and CW21 and the SM6 and H17 sites shewn in Figure 6 and the PKDl gene is thereby absent, the deletion lying prOximally between S 6 and H13; (e) [461] 18bp are deleted in the 75bp intron amplified by the primer pair 3A3C insert at position 3696 of the 3' sequence as shown in Figure 11; (f) [0X1054] 20bp are deleted in the 75bp intron amplified by the primer pair 3A3C insert at position 3696 of the 3' sequence as shown in Figure 11; (g) [WS212] about 75kb are deleted between S 9-CW9 distally and the PKDl 3'UTR proximally as shown in Figure 12; (h) [WS-215] about 160kb are deleted between CW20 and S 6-JH17 as shown in Figure 12; (i) [WS-227] about 50kb are deleted between CW20 and JH11 as shown in Figure 12; (j) [WS-219] about 27kb are deleted between JH1 and H6 as shown in Figure 12; (k) [WS-250] about 160kb are deleted between CW20 and BLu24 as shown in Figure 12; (1) [WS-194] about 65kb is deleted between CW20 and CWIO. T-e invention therefore extends to RNA molecules coπprisir-g an RNA sequence correspcinding to any of the DNA sequences set out above. The molecule is preferably the transcript reference PBP and identifiable from the restriction map of Figure 3a and having a sequence of about 14 Kb. In another aspect, the invention provides a nucleic acid probe having a sequence as set out above; in particular, this invention extends to a purified nucleic acid probe which hybridises to at least a portion of the DNA or RNA molecule of any of the preceding sequences. Preferably, the probe includes a label such as a radiolabel for example a 3 P label. In another aspect, this invention provides a purified DNA or RNA codinq for a protein comprising the amino acid sequence of Figure 7 and/or 10, or a protein polypeptide having homologous properties with said protein, or having at least one functional domain or active site in common with said protein. The DNA molecule defined above may be incorporated in a recombinant cloning vector for expressing a protein having the amino acid sequence of Figure 7 and/or 10, or a protein or a polypeptide having at least one functional domain or active site in common with said protein. In another aspect, the invention provides a polypeptide encoded by a sequence as set out above, or having the amino acid sequence according to the partial amino acid sequence of Figure 7 and/or 10, or a protein or polypeptide having homologous properties with said protein, or having at least one functional domain or active site in common with said protein. In particular, there is provided an isolated, purified or recombinant polypeptide comprising a PKDl protein or a mutant or variant thereof or encoded by a sequence set out above or a variant thereof having substantially the same activity as the PKDl protein. This invention also provides an in vitro method of determining whether an individual is likely to be affected with tuberous sclerosis, comprising the steps of: assaying a sample from the individual to determine the presence and/or amount of PKDl protein or polypeptide having the amino acid sequence of Figure 7 and/or 10. Additionally or alternatively, a sample may be assayed to determine the presence and/or amount of mRNA coding for the protein or polypeptide having the amino acid sequence of Figure 7 and/or 10, or to determine the fragment lengths of fragments of nucleotide sequences coding for the protein or polypeptide of Figure 7 and/or 10, or to detect inactivating mutations in DNA coding for a protein having the amino acid sequence of Figure 7 and/or 10 or a protein having homologous properties. Said screening preferably includes applying a nucleic acid amplification process to said sample to amplify a fragment of the DNA sequence. Said nucleic acid amplification process advantagously utilizes at least one of the following sets of primers as identified herein :- AH3 F9 AH3 B7 3A3 CI 3A3 C2 AH4 F2 JH14 B3 Alternatively, said screening method may comprise digesting said sample to provide EcoRl fragments and hybridising with a DNA probe which hybridises to the EcoRl fragment identified (A) in Figure 3(a), and said DNA probe may comprise the DNA probe CW10 identified herein. Another screening method may comprise digesting said sample to provide BamHI fragments and hybridising with a DNA probe which hybridises to the BamHI fragment identified (B) in Figure 3 (a), and said DNA probe may comprise the DNA probe 1A1H.6 identified herein. A method according to the present invention may comprise detecting a PKDl-associated disorder in a patient suspected of having or having predisposition to, said disorder, the method comprising detecting the presence of and/or evaluating the characteristics of PKDl DNA, PKDl mRNA and/or PKDl protein in a sample taken from the patient. Such method may comprise detecting and/or evaluating whether the PKDl DNA is deleted, missing, mutated, aberrant or not expressing normal PKDl protein. One way of carrying out such a method comprises: A. taking a biological, tissue or biopsy sample from the patient; B. detecting the presence of and/or evaluating the characteristics of PKDl DNA, PKDl mRNA and/or PKDl protein in the sample to obtain a first set of results; C. comparing the first set of results with a second set of results obtained using the same or similar methodology for an individual not suspected of having said disorders; and if the first and second sets of results differ in that the PKDl DNA is deleted, missing, aberrant, mutated or not expressing PKDl protein then that indicates the presence, predisposition or tendency of the patient to develop said disorders. A specific method according to the invention comprises extracting a sample of PKDl DNA or DNA from the PKDl locus purporting to be PKDl DNA from a patient, cultivating the sample in . vitro and analysing the resulting protein, and comparing the resulting protein with normal PKDl protein according to the well- established Protein Truncation Test. Less sensitive tests include analysis of RNA using RT PCR (reverse transcriptase polymerase chain reaction) and examination of genomic DNA. On the other hand, if step C of the method is replaced by: C. comparing the first set of results with a second set of results obtained using the same or similar methodology in an individual known to have the or at least one of said disorder(s); and if the first and second sets of results are substantially identical, this indicates that the PKDl DNA in the patient is deleted, mutated or not expressing normal PKDl protein. The invention further provides a method of characterising a mutation in a subject suspected of having a mutation in the PKDl gene, which method comprises: A. amplifying each of the exons in the PKDl gene of the subject; B. denaturing the complementary strands of the amplified exons; C. diluting the denatured separate, complementary strands to allow each single-stranded DNA molecule to assume a secondary structural conformation; D. subjecting the DNA molecule to electrophoresis under non-denaturing conditions; E. comparing the electrophoresis pattern of the single-stranded molecule with the electrophoresis pattern of a single-stranded molecule containing the same amplified exon from a control individual which has either a normal or PKDl heterozygous genotype; and F. sequencing any amplification product which has an electrophoretic pattern different from the pattern obtained from the DNA of the control individual. The invention also extends to a diagnostic kit for carrying out a method as set out above, comprising nucleic acid primers for amplifying a fragment of the DNA or RNA sequences defined above. The nucleic a ' cicl primers may comprise at least one of the following sets: AH3 F9 : AH3 B7 3A3 CI : 3A3 C2 AH4 F2 : JH14 B3 Another embodiment of kit may combine one or more substances for digesting a sample to provide EcoRl fragments and a DNA probe as previously defined. A further embodiment of kit may comprise one or more substances for digesting a sample to provide BamHI fragments and a DNA probe as previously defined. Still further, a kit may include a nucleic acid probe capable of hybridising to the DNA or RNA molecule previously defined. A vector (such as Bluscript (available from Stratagene)) comprising a nucleic acid sequence set out above; and a host cell (such as E. coli strain SL-1 Blue (available from Stratagene) ) transfected or transformed with the vector are also provided, together with the use of such a vector or a nucleic acid sequence set out above in gene therapy and/or in the preparation of an agent for treating or preventing a PKDl-associated disorder. Therefore there is further provided a method of treating or preventing a PKDl- associated disorder which method comprises administering to a patient in need thereof a functional PKDl gene to affected cells in a manner that permits expression of PKDl protein therein and/or a transcript produced from a mutated chromosome (such as the deleted WS-212 chromosome) which is capable of expressing functional PKDl protein therein. The invention also extends to any inventive combination of features set out a.bove or in the following description. Brief Description Of The Drawings Figure la (top) : A long range map of the terminal region of the short arm of chromosome 16 showing the PKDl candidate region defined by genetic linkage analysis. The positions of selected DNA probes and 5 microsatellites used for haplotype, lindage or heterozygosity analyses are indicated. Markers previously described in linkage disequilibrium studies are shown in bold (from: Harris, et al. , 1990; Harris, et al., 1991; Germino, et al . , 1992; Somlo, et al . , 10 1992; Peral, et al., 1994; Snarey, et al., 1994). (bottom) : A detailed map of the distal part of the PKDl candidate region showing: the area of 16pl3.3 duplicated in 16pl3.1 (hatched); C, Cla I restriction sites; the breakpoints in the somatic cell hybrids, N- 15 OH1 and P-MWH2A; DNA probes and the TSC2 gene. The limits of the position of the translocation breakpoint found in family 77 (see b), determined by evidence of heterozygosity (in 77-4) and PFGE (see c and text) is also indicated. The contig covering the 77 breakpoint 0 region consists of the cosmids: 1, CW9D; 2, ZDS5; 3, JH2A; 4, REP59; 5, JC10.2B; 6, CW10III; 7, SM25A; 8, SMII; 9, NM17. Figure lb: Pedigree of family 77 which segregates a 16;22 translocation; showing the chromosomal 5 composition of each subject. Individuals 77-2 and 77-3 have the balanced products of the exchange - and have PKDl; 77-4 is monosomic for 16pl3.3-- > 16pter and 22qll.21—>22pter - and has TSC. Figure lc: PFGE of DNA from members of the 77 0 family: 77-1 (1) ; 77-2 (2); 77-3 (3) ; 77-4 (4); digested with Cla I and hybridised with SM6. In addition to the normal fragments of 340 and partially digested fragment of 480 kb a proximal breakpoint fragment of approximately joo kb (arrowed) is seen in 5 individua s , 77 2, 77-3ϊ and 77-4; concordant with segregation of the der(16) chromosome. Figure 2: FISH of the cosmid CW10III (cosmid 6; Figure la) to a normal male metaphase. Duplication of this locus is illustrated with two sites of hybridisation on 16p; the distal site (the PKDl region) is arrowed. The signal from the proximal site (16pl3.1) is stronger than that from the distal, indicating that sequences homologous to CW10III are re¬ iterated in 16pl3.1. Figure 3a: A detailed map of the 77 translocation region showing the precise localisation of the 77 breakpoint and the region that is duplicated in 16pl3.1 (hatched). DNA probes (open boxes); the transcripts, PKDl and TSC2 (filled boxes; with direction of transcription indicated by an arrow) and cDNAs (grey boxes) are shown below the genomic map. The known genomic extent of each gene is indicated at the bottom of the diagram and the approximate genomic locations of each cDNA is indicated under the genomic map. The positions of genomic deletions found in PKDl patients, 0X875 and 0X114, are also indicated. Restriction sites for EcoR I (E) and incomplete maps for BamH I (B); Sac I (S) and Xba I (X) are shown. SM3 is a 2kb BamHI fragment shown at the 5' end of the gene. Figure 3b: Southern blots of BamH I digested DNA from individuals: 77-1 (1); 77-2 (2); and 77-4 (4) hybridised with: left panel, 8S3 and right panel, 8S1 (see a). 8S3 detects a novel fragment on the telomeric side of the breakpoint (12 kb: arrowed) associated with the der(22) chromosome in 77-2, but not 77-4; 8S1 identifies a novel fragment on the centromeric side of the breakpoint (9 kb: arrowed) - associated with the der(16) chromosome - in 77-2 and 77-4. The telomeric breakpoint fragment is also seen weakly with 8S1 (arrowed) indicating that the breakpoint lies in the distal part of 8S1. The 3S3 and 8S1 loci are both duplicated; the normal BamH I fragment detected at the 16pl3.3 site by these probes is 11 kb (see a), but a similar sized fragment is also detected at the 16pl3.1 site. Consequently, the breakpoint fragments are much fainter than the normal (16pl3.1 plus 16pl3.3) band. Figure 4a: PBP cDNA, 3A3, hybridised to a Northern blot containing " 1 mg polyA selected mRNA per lane of the tissue specific cell lines: lane 1, MJ, EBV- transformed lymphocytes; lane 2, K562, erythroleukaemia; lane 3, FS1, normal fibroblasts; lane 4, HeLa, cervical carcinoma; lane 5, G401, renal Wilm's tumour; lane 6, Hep3B, hepatoma; lane 7, HT29, colonic adenocarcino a; lane 8, SW13, adrenal carcinoma; lane 9, G-CCM, astrocytoma. A single transcript of approximately 14 kb is seen; the highest level of expression is in fibroblasts and in the astrocytoma cell line, G-CCM. Although in this comparative experiment little expression is seen in lanes 1, 4 and 7, we have demonstrated at least a low level of expression in these cell lines on other Northern blots and by RT-PCR (see later). Figure 4b: A Northern blot containing ~ 20 mg of total RNA from the cell line G-CCM hybridised with cDNAs or a genomic probe which identify various parts of the PBP gene. Left panel, a single " 14 kb transcript is seen with a cDNA from the single copy area, 3A3. Right panel, a cDNA, 21P.9, that is homologous to parts of the region that is duplicated (JH12, JH8 and JH10; see Figure 3a) hybridises to the PBP transcript and three novel transcripts; HG-A ( ~ 21 kb), HG-B ( " 17 kb) and HG-C (8.5 kb) . A similar pattern of transcripts is seen with cDNAs and genomic fragments that hybridise to the area between JH5 and JH13, with the exception of the JH8 area. Middle panel, JH8 hybridises to the transcripts PBP, HG-A and HG-B but not to HG-C. ' ' ^ - - Figure 4c: A Northern blot of 20mg total fibroblast RNA from: normal control (N); 77-2 (2); 77-4 (4) hybridised with 8S1, which contains the 16;22 translocation breakpoint (see Figure 3). A transcript of ~ 9 kb (PBP-77) is identified in the two patients with this translocation but not in the normal control. PBP-77 is a chimeric PBP transcript formed due to the translocation and is not seen in 77-2 or 77-4 RNA with probes which map distal to the breakpoint. Figure 5a: FIGE of DNA from: normal (N) and ADPKD patient 0X875 (875) , digested with EcoR I and hybridised with, left panel, CWIO; middle panel, JH1. Normal fragments of 41 kb (plus a 31 kb fragment from the 16pl3.1 site), CWIO, and 18 kb, JHI, are identified with these probes; 0X875 has an additional 53 kb band (arrowed) . The EcoR I site separating these two fragments is removed by the deletion (see Figure 3a). The right panel shows a Southern blot of BamH I digested DNA (as above) hybridised with 1A1H.6. A novel fragment of 9.5 kb is seen in 0X875 DNA, as well as the normal 15 kb fragment. These results indicate that 0X875 has a 5.5 kb deletion; its position was determined more precisely by mapping relative to two Xba I sites which flank the deletion (see figure 3a). Figure 5b: Northern blot of total fibroblast RNA, as (a), hybridised with the cDNAs, AH4, 3A3 and AH3. A novel transcript (PBP-875) of ~ 11 kb is seen with AH4 (the band is reduced in intensity because the probe is partly deleted) and AH3 (arrowed), which flank the deletion, but not 3A3 which is entirely deleted (see figure 3a). The transcripts HG-A, HG-B and HG-C, from the duplicated area, are seen with AH3 (see figure 4b). Figure 5c: Left panel; FIGE of DNA from: normal (N) and ADPKD patient 0X114 (114), digested with EcoR I and hybridised with CWIO; a novel fragment of 39 kb (arrowed) ± s seen in 0X114. Middle panel; DNA, as above, plus the normal mother (M) and brother (B) of 0X114 digested with BamH I and hybridised with CW21. A larger than normal fragment of 19 kb (arrowed) was detected in 0X114 but not other family members due to deletion of a BamH I site; together these results are consistent with a 2 kb deletion (see Figure 3a). Right panel; RT-PCR of RNA, as above, with primers flanking the 0X114 deletion (see Experimental Procedures). A novel fragment of 810 bp (arrowed) is seen in 0X114, indicating a deletion of 446 bp in the PBP transcript. Figure 5d: RT-PCR of RNA from: ADPKD patient 0X32 (32) plus the probands, normal mother (M) and affected father (F) and sibs (1) and (2) using the C primer pair from 3A3 (see Experimental Procedures) . A novel fragment of 125 bp is detected in each of the affected individuals . Figure 6: Map of the region containing the TSC2 and PBP genes showing the area deleted in patient WS-53 and the position of the 77 translocation breakpoint. Localisation of the distal end of the WS-53 deletion was previously described (European Chromosome 16 Tuberous Sclerosis Consortium, 1993) and we have now localised the proximal end between SM6 and JHI7. The size of the aberrant Mlu I fragment in WS-53, detected by JH1 and JH17, is 90kb and these probes lie on adjacent Mlu I fragments of 120kb and 70kb, respectively. Therefore the WS-53 deletion is ~ lOOkb. Restriction sites for: Mlu I (M); Nru I (R); Not I (N); and partial maps for Sac II (S) and BssH II (H) are shown. DNA probes (open boxes) and the TSC2 and PBP transcripts (filled boxes) are indicated below the line with their known genomic extents (brackets) . The locations of the microsatellites KG8 and SM6 are also indicated. Figure 7:. -The partial nucleotide sequence (cDNA) of the PKDl transcript extending 563lbp to i-he 3' end of the gene. The corresponding predicted protein (also shown in SEQ ID NO: 4:) is shown below the sequence and extends from the start of the nucleotide sequence. The GT-repeat, KG8, is in the 3' untranslated region between 5430-5448 bp. This sequence corresponds to GenBank Accession No. L33243 and is shown in SEQ ID NO: 3: . Figure 8: The sequence of the probe 1A1H0.6 (also shown in SEQ ID NO: 5:). Figure 9: The sequence (SEQ ID NO: 6: ) of the probe CWIO which is about 0.5kb. Figure 10: The larger partial nucleotide sequence (SEQ ID NO: 1:) of the PKDl transcript (cDNA) extending from bp 2 to 13807bp to the 3' end of the gene together with the corresponding predicted protein (also shown in SEQ ID NO: 2: ) . This larger partial sequence encompasses the (smaller) partial sequence of Figure 7 from amino acid no. 2726 in SEQ ID NO: 3: and relates to the entire PKDl gene sequence apart from its extreme 5' end. Figure 11: A map of the 75bp intron amplified by the primer set 3A3C insert at position 3696 of the 3' sequence showing the positions of genomic deletions found in PKDl patients 461 and OX1054. Figure 12: A map of the region of chromosome 16 containing the TSC2 and PKDl genes showing the areas affected in patients WS-215, WS-250, WS-212, WS-194, WS-227 and WS-219; also WS-53 (but cf. Figure 6) . Genomic sites for the enzymes Mlul (M), Clal (C), Pvul (P) and Nrul (R) are shown. Positions of single copy probes and cosmids used to screen for deletions are shown below the line which represents ~400kb of genomic DNA. The genomic distribution of the approximately 45kb TSC2 gene and known extent of the PKDl gene are indicated above. The hatched area re resents an ~50kb region which is duplicated more proximally on chromosome 16p. Detailed Description of the Drawings A translocation associated with ADPKD A major pointer to the identity of the PKDl gene was provided by a Portuguese pedigree (family 77) with both ADPKD and TSC (Figure lb) . Cytogenetic analysis showed that the mother, 77-2, has a balanced translocation, 46XX t ( 16; 22 ) (pl3.3;qll .21 ) which was inherited by her daughter, 77-3. The son, 77-4, has the unbalanced karyotype, 45XY-16-22+der ( 16 ) ( 16qter — > 16pl3.3: : 22qll .21-- >2qter ) and consequently is mono¬ somic for 16pl3.3 — >16pter as well as for 22qll.21 — > 22pter. This individual has the clinical phenotype of TSC (see Experimental Procedures); the most likely explanation is that the TSC2 locus located within 16pl3.3 is deleted in the unbalanced karyotype. Further analysis revealed that the mother (77-2), and the daughter (77-3) with the balanced translocation, have the clinical features of ADPKD (see Experimental Procedures), while the parents of 77-2 were cytogenetically normal, with no clinical features of TSC and no renal cysts on ultrasound examination (aged 67 and 82 years). Although kidney cysts can be a feature of TSC, no other clinical signs of TSC were identified in 77-2 or 77-3, making it unlikely that the polycystic kidneys were due to TSC. We therefore investigated the possibility that the translocation disrupted the PKDl locus in 16pl3.3 and proceeded to identify and clone the region containing the breakpoint. The 77 family was analysed with polymorphic markers from 16pl3.3. Individual 77-4 was hemizygous for MS205.2 and GGGl, but heterozygous for SM6 and more proximal markers, locating the translocation breakpoint between GGGl and SM6 (see Figure la). Fluorescence in situ hybridisation (FISH) of a cosmid from the TSC2 region, CW9D (cosmid 1 in Figure la), to metaphase spreads showed that it hybridised to the der(22) chromosome of 77-2; placing the breakpoint proximal to CW9D and indicating that 77-4 was hemizygous for this region consistent with his TSC phenotype. DNA from members of the 77 family was digested with Cla I, separated by PFGE and hybridised with SM6; revealing a breakpoint fragment of ~ 100 kb in individuals with the der(16) chromosome (Figure lc). The small size of this novel fragment enabled the breakpoint to be localised distal to SM6 in a region of just 60 kb (Figure la). A cosmid contig covering this region was therefore constructed (see Experimental Procedures for details). The translocation breakpoint lies within a region duplicated elsewhere on chromosome 16p (16pl3.1) It was previously noted that the region between CW21 and N54 (Figure la) was duplicated at a more proximal site on the short arm of chromosome 16 (Germino, et al. , 1992; European Chromosome 16 Tuberous Sclerosis Consortium, 1993). Figure 2 shows that a cosmid, CWIOIII, from the duplicated region hybridises to two points on 16p; the distal, PKDl region and a proximal site positioned in 16pl3.1. The structure of the duplicated area is complex with each fragment present once in-16pl3.3 re-iterated two-four times in 16pl3.1 (see Figure 2). Cosmids spanning the duplicated area in 16pl3.3 were subcloned (see Figure 3a and Experimental Procedures for details) and a restriction map was generated. A genomic map of the PKDl region was constructed using a radiation hybrid, Hyl45.19 which contains the distal portion of 16p but not the duplicate site in 16pl3.1. To localise the 77 translocation breakpoint, subclones from the target region were hybridised to 77- 2 DNA, digested with Cla I and separated by PFGE. Once probes mapping across the breakpoint were identified they were hybridised to conventional Southern blots of 77 family DNA. Figure 3b shows that novel BamH I fragments were detected from the centromeric and telomeric side of the breakpoint, which was localised to the distal part of the probe 8S1 (Figure 3a) . Hence, the balanced translocation was not associated with a substantial deletion, and the breakpoint was located more than 20 kb proximal to the TSC2 locus (Figure 3a) . These results supported the hypothesis that polycystic kidney disease in individuals with the balanced translocation (77-2 and 77-3) was not due to disruption of the TSC2 gene, but indicated that a separate gene mapping just proximal to TSC2, was likely to be the PKDl gene. The polycystic breakpoint (PBP) gene is disrupted by the translocation Localisation of the 77 breakpoint identified a precise region in which to look for a candidate for the PKDl gene. During the search for the TSC2 gene we identified other transcripts not associated with TSC including a large transcript (~ 14 kb) partially represented in the cDNAs 3A3 and AH4 which mapped to the genomic fragments CW23 and CW21 (Figure 3a). The orientation of the gene encoding this transcript had been determined by the identification of a polyA tract in the cDNA, AH4 : the 3' end of this gene lies very close to the TSC gene, in a tail to tail orientation (European Chromosome 16 Tuberous Sclerosis Consortium, 1993) . To determine whether this gene crossed the translocation breakpoint genomic probes from within the duplicated area and flanking the breakpoint were hybridised to Northern blots. Probes from both sides of the breakpoint, between JH5 and JH13 identified the 14 kb transcript (Figure 3a and see below for details). Therefore, this gene previously called 3A3 , but now designated the PBP gene extended over the 77 breakpoint and consequently was a candidate for the PKDl gene. A walk was initiated to increase the extent of the PBP cDNA contig and several new cDNAs were identified using probes from the single copy (non- duplicated) region (see Experimental Procedures for details). A cDNA contig was constructed which extended " 5.7 kb, including ~ 2 kb into the area that is duplicated (Figure 3a) . Expression of the PBP gene Initial studies of the expression pattern of the PBP gene were undertaken with cDNAs that map entirely within the single copy region (e.g. AH4 and 3A3 ) . Figure 4a shows that the ~ 14 kb transcript was identified by 3A3 in various tissue-specific cell lines. From this and other Northern blots we concluded that the PBP gene was expressed in all of the cell lines tested, although often at a low level. The two cell lines which showed the highest level of expression were fibroblasts and a cell line derived from an astrocytoma, G-CCM. Significant levels of expression were also obtained in cell lines derived from kidney (G401) and liver (Hep3B). Measuring the expression of the PBP gene in tissue samples by Northern blotting proved difficult because such a large transcript is susceptable to minor RNA degradation. However, initial results with an RNAse protection assay, using a region of the gene located in the single copy area (see Experimental Procedures), showed a moderate level of expression of the PBP gene in tissue obtained from normal and polycystic kidney (data not shown). The widespread expression of the PBP gene is consistent with the systemic nature of ADPKD. Identi ication of transcripts that are partially homologous to the PBP transcript New cDNAs were identified with the genomic fragments, JH4 and JH8, that map to the duplicated region (Figure 3a and see Experimental Procedures). However, when these cDNAs were hybridised to Northern blots a more complex pattern than that seen with 3A3 was observed. As well as the "14 kb PBP transcript, three other, partially homologous transcripts were identified designated homologous gene-A (HG-A; ~ 21 kb), HG-B ( " 17 kb ) and HG-C (8.5 kb ) (Figure 4b). There were two possible explanations for these results, either the HG transcripts were alternatively spliced forms of the PBP gene, or the HG transcripts were encoded by genes located in 16pl3.1. To determine the genomic location of the HG loci a fragment from the 3 ' end of one HG cDNA (HG-4/1.1) was isolated. HG-4/1.1 hybridised to all three HG transcripts, but not to the PBP transcript and on a hybrid panel it mapped to 16pl3.1 (not the PKDl area). These results show that all the HG transcripts are related to each other outside the region of homology with the PBP transcript and that the HG loci map to the proximal site (16pl3.1) . An abnormal transcript associated with the 77 translocation As the PBP gene was transcribed across the region disrupted by the 77 translocation breakpoint, in a proximal to distal direction on the chromosome (see Figure 3a) it was possible that a novel transcript originating from the PBP promotor would be found in this family. Figure 4c shows that using a probe to the PBP transcript that mapped mainly proximal to the breakpoint, a novel transcript of approximately 9 kb (PPP-77) derived from the der(16) product of the translocation was detected. Interestingly, the PBP-77 transcript appears to be expressed at a higher level than the normal PBP product. These results confirmed that the 77 translocation disrupts the PBP gene and supports the hypothesis that this is the PKDl gene. Mutations of the PBP gene in other ADPKD patients To prove that the PBP gene is the defective gene at the PKDl locus, we analysed this region for mutations in patients with typical ADPKD. The 3' end of the PBP gene was most accessible to study as it maps outside the duplicated area. To screen this region BamH I digests of DNA from 282 apparently unrelated ADPKD patients were hybridised with the probe 1A1H.6, (see Figure 3a). In addition, a large EcoR I fragment (41 kb) which contains a significant proportion of the PBP gene was assayed by field inversion gel electrophoresis (FIGE) in 167 ADPKD patients, using the probe CWIO. Two genomic rearrangements were identified in ADPKD patients by these procedures; each identified by both methods . The first rearrangement was identified in patient 0X875 (see Experimental Procedures for clinical details) who was shown to have a 5.5 kb genomic deletion within the 3' end of the PBP gene, producing a smaller transcript (PBP-875) (see Figures 5a, b and 3a for details). This genomic deletion results in a " 3 kb internal deletion of the transcript with the " 500 bp adjacent to the polyA tail intact. In this family linkage of ADPKD to chromosome 16 could not be proven because although 0X875 has a positive family history of ADPKD there were no living, affected relatives. However, paraffin-embedded tissL rom her affected father (now deceased) was available. We demonstrated that this individual had the same rearrangement as 0X875 by PCR amplification of a 220bp fragment spanning the deletion (data not shown) . This result and analysis of two unaffected sibs of 0X875, that did not have the deletion, showed that this mutation was transmitted with ADPKD. The second rearrangement detected by hybridisation was a 2 kb genomic deletion within the PBP gene, in ADPKD patient 0X114 (see Experimental Procedures for clinical details and Figures 5c and 3a). No abnormal PBP transcript was identified by Northern blot analysis, but using primers flanking the deletion (see Experimental Procedures) a shortened product was detected by RT-PCR (Figure 5c). This was cloned and sequenced and shown to have a frame-shift deletion of 446 bp (between base pair 1746 and 2192 of the sequence shown in Figure 7). 0X114 is the only member of the family with ADPKD (she has no children) and ultrasound analysis of her parents at age 78 (father) and 73 years old (mother) showed no evidence of renal cysts. Somatic cell hybrids were produced from 0X114 and the deleted chromosome was found to be of paternal origin by haplotype analysis. The father of 0X114 is now deceased but analysis of DNA from the brother of 0X114 (0X984) with seven microsatellite markers from the PKDl region (see Experimental Procedures) showed that he shares the same paternal chromosome, in the PKDl region, as 0X114. Renal ultrasound revealed no cysts in 0X984 at age 53 and no deletion was detected by DNA analysis (Figure 5c). Hence, the deletion in 0X114 is a de novo event associated with the development of ADPKD. Although it is not possible to show that the ADPKD is chromosome 16-linked, the location of the PBP gene indicates that this is a de novo PKDl mutation. To identify more PKDl associated mutations, single copy regions of the PBP gene were analysed by RT-PCR using RNA isolated from lymphoblastoid cell lines established from ADPKD patients. cDNA from 48 unrelated patients was amplified with the primer pair 3A3 C (see Experimental Procedures) and the product of 260 bp was analysed on an agarose gel. In one patient, 0X32, an additional smaller product (125 bp) was identified, consistent with a deletion or splicing mutation. 0X32 comes from a large family in which the disease can be traced through three generations . Analysis of RNA from two affected sibs of 0X32 and his parents showed that the abnormal transcript segregates with PKDl (Figure 5d) . Amplification of normal genomic DNA with the 3A3 C primers generates a product of 418 bp; sequencing showed that this region contains two small introns (5', 75 bp and 3', 83 bp ) flanking a 135 bp exon. The product amplified from 0X32 genomic DNA was normal in size, excluding a genomic deletion. However, heteroduplex analysis of that DNA revealed larger heteroduplex bands, consistent with a mutation within that genomic interval. The abnormal 0X32, RT-PCR product was cloned and sequenced: this demonstrated that, although present in genomic DNA, the 135 bp exon was missing from the abnormal transcript. Sequencing of 0X32 genomic DNA demonstrated a G — >C transition at +1 of the splice donor site following the 135 bp exon. This mutation was confirmed in all available affected family members by digesting amplified genomic DNA with the enzyme Bst NI : a site is destroyed by the base substitution. The splicing defect results in an in- frame deletion of 135 bp from the PBP transcript (3696 bp to 3831 bp of the sequence shown in Figure 7). Together, the three intragenic mutations confirm that the PBP gene is the defective gene at the PKDl locus. Deletions that disrupt the TSC2 and the PKDl gene We previous!' identified a deletion (WS-53) which disrupts the TSC2 gene and the PKDl gene (European Chromosome 16 Tuberous Sclerosis Consortium, 1993), although its full proximal extent was not determined. Further study has shown that the deletion extends ~ 100 kb (see Figure 6 for details) and deletes most if not all of the PKDl gene. This patient has TSC but also has unusually severe polycystic disease of the kidneys. Other patients with a similar phenotype have also been under investigation. Deletions involving both TSC2 and PKDl were identified and characterised in six patients in whom TSC was associated with infantile polycystic kidney disease. As well as the deletion in WS-53, those in WS-215 and "S-250 also extended proximally well beyond the known distribution of PKDl and probably delete the entire gene. The deletion in WS-194 extended over the known extend of PKDl, but not much further proximally, while the proximal breakpoints in WS-219 and WS-227 lay within PKDl itself. Northern analysis of case WS-219 with probe JH8, which lies outside the deletion, showed a reduced level of the PKDl transcript but no evidence of an abnormally sized transcript (data now shown) . Analysis of samples from the clinically unaffected parents of patients WS-53, WS-215, WS-219, WS-227 and WS-250 showed the deletions in these patients to be de novo. The father of WS-194 was unavailable for study. In a further case (WS-212), renal ultrasound showed no cysts at four years of age but a deletion was identified which removed the entire TSC2 gene and ' deleted an Xbal site which is located 42bp 5 ' to the polyadenylation signal of PKDl. To determine the precise position of the proximal breakpoint in PKDl, a 587bp probe from the 3' untranslated region (3'UTRP) was hybridised to Xbal digested DNA. A 15kb XbaL breakpoint fragment was detected with an approximately equal intensity to the normal fragment of 6kb, indicating that most of the PKD13'UTR was preserved on the mutant chromosome. Evidence that a PKDl transcript is produced from the deleted chromosome in WS-212 was obtained by 3' rapid identification of cDNA ends (RACE) with a novel, smaller product generated from WS-212 cNDA. Characterisation of this product showed that polyadenylation occurs 546bp 5' to the normal position, within the 3'UTR of PKDl (231bp 3' to the stop codon at 5073bp of the described PKDl sequence 14 ) . A transcript with an intact open reading frame is thus produced from the deleted WS-212 chromosome. It is likely that a functional PKDl protein in produced from this transcript, explaining the lack of cystic disease in this patient. The sequence preceeding the novel site of polyA addition is: AGTCAGTAATTTATATGGTGTTAAAATGTG(A)n. Although not conforming precisely to the concensus of AATAAA, it is likely that part of this AT rich region acts as an alternative polyadenylation signal if, as in this case, the normal signal is deleted (a possible sequence is underlined) . The WS-212 deletion if 75kb between SM9-CW9 distally and the PKDl 3 'UTR proximally. The WS-215 deletion is 160kb between CW15 and SM6-JH17. WS-194 has 65kb deleted between CW20 and CW10-CW36. WS-227 has a 50kb deletion between CW20 and JH11 and WS-219 •has a 27kb deletion between JHl and JH6. The distal end of the WS-250 deletion is in CW20 but the precise location of the proximal end is not known. However, the same breakpoint fragment of 320kb is seen with Pvul-digested DNA using probes on adjacent Pvul fragments, CE18 (which normally detects a 245kb fragment) and BLu24 (235kb). Hence this deletion can be estimated ~160kb. b. PFGE analysis of the deletion in WS-219. Mlul digested DNA from a normal control (N) and WS-219 probed with the clones H2 , JHl, CW21 and CWIO which detect an ~130kb fragment in normal individuals. CWIO also detects a much smaller fragment froπi the duplicated region situated more proximally on 16p. A novel fragment of ~ 100kb is seen in WS-219 with probes H2 and CWIO which flank the deletion in this patient. JHl is partially deleted but detects the novel band weakly. The aberrant fragment is not detected by CW-21, which is deleted on the mutant chromosome. BamHI digested DNA of normal control (N) and WS-219 separated by conventional gel electrophoresis and hybridised to probes JHl and JH6 which flank the deletion. The same breakpoint fragment of " 3kb is seen with both probes, consistent with a deletion of ~27kb ending within the BamHI fragments seen by these probes. Two further deletions In addition we have characterised two further mutations of this gene which were identified in typical PKDl families. In both cases the mutation is a deletion in the 75bp intron amplified by the primer pair 3A3C (European Polycystic Kidney Disease Consortium, 1994). The deletions are of 18bp and 20bp, respectively, in the patients 461 and OX1054. Although these deletions do not disrupt the highly conserved sequences flanking the exon/intron boundaries, they do result in aberrant splicing of the transcript. In both cases, two abnormal mRNAs are produced, one larger and one smaller than normal. Sequencing of these cDNAs showed that the larger transcript includes the deleted intron, and so has an in-frame insertion of 57bp in 461, while OX1054 has a frameshift insertion of 55bp. The smaller transcript is due to activation of a cryptic splice site in the exon preceding the deleted intron and results in an in-frame deletion of 66bp in both patients. The demonstration of two additional mutations of this gene in PKDl patients further confirms that this is the PKDl gene. Characterisation of the PKDl gene To characterise the PKDl gene further, evolutionary consexvatlon was analys e ~ " by" '-zo . c blotting' . Using probes from the single copy, 3' region ( 3A3) and from the duplicated area (JH4, JH8 ) the PKDl gene was conserved in other mammalian species, including horse, dog, pig and rodents (data not shown). No evidence of related sequences were seen in chicken, frog or drosophila by hybridisation at normal stringency. The degree of conservation was similar when probes from the single copy or the duplicated region were employed. The full genomic extent of the PKDl gene is not yet known, although results obtained by hybridisation to Northern blots show that it extends from at least as far as JH13. Several CpG islands have been localised 5' of the known extent of the PKDl gene (Figure 6), although there is no direct evidence that any of these are associated with this gene. The cDNA contig extending 5631 bp to the 3' end of the PKDl transcript was sequenced; where possible more than one cDNA was analysed and in all regions both strands were sequenced (Figure 7). We estimated that this accounts for " 40% of the PKDl transcript. An open reading frame was detected which runs from the 5 ' end of the region sequenced and spans 4842 bp, leaving a 3' untranslated region of 789 bp which contains the previously described microsatellite, KG8 (Peral, et al . , 1994; Snarey, et al . , 1994). A polyadenylation signal is present at nucleotides 5598-5603 and a polyA tail was detected in two independent cDNAs (AH4 and AH6) at position, 5620. Comparison with the cDNAs HG- 4 and 11BHS21, which are encoded by genes in the duplicate, 16pl3.1 region, show that 1866 bp at the 5' end of the partial PKDl sequence shown in Figure 7 lies within the duplicated area. The predicted amino acid sequence from the available open reading frame extends 1614 residues, and ΪP shown in Figure 7. A search of the swiccprot and NBRF data bases with the available protein sequence, using the Blast programme (Altschul, et al. , 1990) identified only short regions of similarity (notably, between amino-acids 690-770 and 1390-1530) to a diverse group of proteins; no highly significant areas of homology were recognised. The importance of the short regions of similarity is unclear as the search for protein motifs with the ProSite Programme did not identify any recognised functional protein domains within the PKDl gene. The task of identifying and characterising the PKDl gene has been more difficult than for other disorders because more than three quarters of the gene is embedded in a region of DNA that is duplicated elsewhere on chromosome 16. This segment of 40-50 kb of DNA, present as a single copy in the PKDl area (16pl3.3), is re-iterated as several divergent copies in the more proximal region, 16pl3.1. This proximal site contains three gene loci (HG-A, -B and -C) that each produce polyadenylated mRNAs and share substantial homology to the PKDl gene; it is not known whether these partially homologous transcripts are translated into functional proteins. Although gene amplification is known as a major mechanism for creating protein diversity during evolution, the discovery of a human disease locus embedded within an area duplicated relatively recently is a new observation. In this case because of the recent nature of the reiteration the whole duplicated genomic region retains a high level of homology, not just the exons. The sequence of events leading to the duplication and which sequence represents the original gene locus are not yet clear. However, early evidence of homology of the 3 ' ends of the three HG transcripts which are different from the 3 ' end of the PKDl gene indicated that the loci in 16pl3.1 have probably ar-i?on by further reiteration of sequences at this site, after it separated from the distal locus. To try to overcome the duplication problem we have employed an exon linking approach using RNA isolated from a radiation hybrid, Hyl45.19, that contains just the PKDl part of chromosome 16, and not the duplicate site in 16pl3.1. Hence, this hybrid produces transcripts from the PKDl gene but not from the homologous genes (HG-A, HG-B and HG-C). We have also sequenced much of the genomic region containing the PKDl gene, from the cosmid JH2A, and have sequenced a number of cDNAs from the HG locus. To determine the likely position of PKDl exons in the genomic DNA we compared HG cDNAs, (HG-4 and HG-7 ) to the genomic sequence. We then designed primers with sequences corresponding to the genomic DNA, to regions identified by the HG exons and employing cDNA generated from the hybrid Hyl45.19, we amplified sections of the PKDl transcript. The polymerase Pfu was used to minimise incorporation errors. These amplified fragments were then cloned and sequenced. The PDK1 cDNA contig whose sequence is shown in Figure 10 is made up of (3 '-5' ) the original 5.7 kb of sequence shown in Figure 7, and the cDNAs : gap α 22 (890 bp), gap gamma (872 bp) , a section of genomic DNA from the clone JH8 (2,724 bp) which corresponds to a large exon, S1-S3 (733 bp), S3- S4 (1,589 bp) and S4-S13 (1,372 bp). Together these make a cDNA of 13,807 bp with the extreme 5' end of the transcript still uncharacterised. When these cDNAs from the PKDl contig were sequenced an open reading frame was found to run from the start of the contig to the previously-identified stop codon, a region of 13,018 bp. The predicted protein encoded by the PKDl transcript is also shown in Figure 10 and has 4,339 amino acid residues. We have therefore compelling evidence that mutations of the PKDl gene give rise to the typical phenotype of ADPKD. The location of this gene within the PKDl candidate region and the available genetic evidence from the families with mutations show that this is the PKDl gene. The present invention therefore includes the PKDl gene itself and the six PKDl- associated mutations which have been described: a de novo translocation, which was subsequently transmitted with the phenotype; two intragenic deletions (one a de novo event); two further deletions; and a splicing defect. It has previously been argued that PKDl could be recessive at the cellular level, with a second somatic mutation required to give rise to cystic epithelium (Reeders, 1992). This "two hit" process is thought to be the mutational mechanism giving rise to several dominant diseases, such as neurofibromatosis (Legius, et al., 1993) and tuberous sclerosis (Green, et al., 1994) which result from a defect in the control of cellular growth. If this were the case, however, we might expect that a proportion of constitutional PKDl mutations would be inactivating deletions as seen in these other disorders. The location of the PKDl mutations may, however, reflect some ascertainment bias as it is this single copy area which has been screened most intensively for mutations. Nevertheless, no additional deletions were detected when a large part of the gene was screened by FIGE, and studies by PFGE showed no large deletions of this area in 75 PKDl patients. It is possible that the mutations detected so far result in the production of an abnormal protein which causes disease through a gain of function. However, it is also possible that these mutations eliminate the production of functional • protein from ^ this- chromosome and result in th PKDi phenotype by haploinsufficiency, or only after loss of the second PKDl homologue by somatic mutation. At least one mutation which seems to delete the entire PKDl gene has been identified (WS-53) but in this case it also disrupts the adjacent TSC2 gene and the resulting phenotype is of TSC with severe cystic kidney disease. Renal cysts are common in TSC so that the phenotypic significance of deletion of the PKDl gene in this case is difficult to assess. It is clear that not all cases of renal cystic disease in TSC are due to disruption of the PKDl gene; chromosome 9 linked TSC (TSC1) families also manifest cystic kidneys and we have analysed many TSC2 patients with kidney cysts who do not have deletion of the PKDl gene. Preliminary analysis of the PKDl protein sequence has highlighted two regions which provide some clues to the possible function of the PKDl gene. At the extreme 5 ' end of the characterised region are two leucine-rich repeats (LRRs) (amino acids 29-74) flanked by characteristic amino flanking (amino acids 6-28) and carboxy flanking sequences (amino acids 76-133) (Rothberg et al, 1990) . LRRs are thought to be involved in protein-protein interations (Kobe and Deisenhofer, 1994) and the flanking sequences are only found in extracellular proteins. Other proteins with LRRs flanked on the amino and carboxy sides are receptors or are involved in adhesion or cellular signalling. Further 3' on the protein (amino acids 350-515) is a C-type lectin domain (Curtis et al, 1992) . This indicates that this region binds carbohydrates and is also likely to be extracellular. These two regions of homology indicate that the 5' part of the PKDl protein is extracellular and involved in protein-protein interactions. It is possible that this protein is a constituent of, or plays a role in assembling, the extracellular matrix (ECM) and may act as an adhesive protein in the ECM. It is also possible that the extracellular portion of this protein is important in signalling to other cells. The function of much of the PKDl protein is still not fully known but the presence of several hydrophobic regions indicates that the protein may be threaded through the cell membrane. Familial studies indicate that de novo mutations probably account for only a small minority of all ADPKD cases; a recent study detected 5 possible new mutations in 209 families (Davies, et al . , 1991). However, in our study one of three intragenic mutations detected was a new mutation and the PKDl associated translocation was also a de novo event. Furthermore, the mutations detected in the two familial cases do not account for a significant proportion of the local PKDl. The 0X875 deletion was only detected in 1 of 282 unrelated cases, and the splicing defect was seen in only 1 of 48 unrelated cases. Nevertheless, studies of linkage disequilibrium have found evidence of common haplotypes associated with PKDl in a proportion of some populations (Peral, et al. , 1994; Snarey, et al . , 1994) suggesting that common mutations will be identified. Once a larger range of mutations have been characterised it will be possible to evaluate whether the type and location of mutation determines disease severity, and if there is a correlation between mutation and extra-renal manifestations. Previous studies have provided some evidence that the risk of cerebral aneurysms 'runs true' in families (Huston, et al. , 1993) and that some PKDl families exhibit a consistently mild phenotype (Ryynanen, et al., 1987). A recent study has concluded that there is evidence of ^ . anticipation in ADPKD families, especially if the disease is transmitted through the mother (Fink, et al., 1994) . Furthermore, analysis of families with early manifestation of ADPKD show that there is a significant intra-familial recurrence risk and that childhood cases are most often transmitted maternally (Fink, et al., 1993; Zerres, et al., 1993) . This pattern of inheritence is reminiscent of that seen in diseases in which an expanded trinucleotide repeat was found to be the mutational mechanism (reviewed in Mandel, 1993). However, no evidence for an expanding repeat correlating with PKDl has been found in this region although such a sequence cannot be excluded. There is ample evidence that early presymptomatic diaqnosis of PKDl is helpful because it allows complications such as hypertension and urinary tract infections to be monitored and treated quickly (Ravine, et al., 1991). The identification of mutations within a family will allow rapid screening of that and other families with the same mutation. However, genetic linkage analysis is likely to remain important for presymptomatic diagnosis. The accuracy and ease of linkage based diagnosis will be improved by the identification of the PKDl gene as a microsatellite lies in the 3' untranslated region of this gene (KG-8) and several CA repeats are located 5' of the gene (see Figure la and 6; Peral, et al. , 1994; Snarey, et al. , 1994) . Experimental Procedures Clinical Details of Patients Family 77 77-2 and 77-3 are 48 and 17 years old, respectively, and have typical ADPKD. Both have bilateral polycystic kidneys and 77-2 has impaired renal function. Neither patient manifests any signs of TSC (apart from cystic kidneys) on clinical and ophthalmological examination or by CT scan of the brain. 77-4 is 13 years old, severely mentally retarded and has multiple signs of TSC including adenoma sebaceum, depigmented macules and periventricular calcification on CT scan. Renal ultrasound reveals a small number of bilateral renal cysts. ADPKD patients 0X875 developed ESRD from ADPKD, aged 46. Progressive decline in renal function had been observed over 17 years; ultrasound examinations documented enlarging polycystic kidneys with less extensive hepatic cystic disease. Both kidneys were removed after renal transplantation and pathological examination showed typical advanced cystic disease in kidneys weighing 1920g and 3450g (normal average 120g) . 0X114 developed ESRD from ADPKD aged 54: diagnosis was made by radiological investigation during an episode of abdominal pain aged 25. A progressive decline in renal function and the development of hypertension was subsequently observed. Ultrasonic examination demonstrated enlarged kidneys with typical cystic disease, with less severe hepatic involvement. 0X32 is a member of a large kindred affected by typical ADPKD in which several members have developed ESRD. The patient himself has been observed for 12 years with progressive renal failure and hypertension following ultrasonic demonstration of polycystic kidneys . No signs of TSC were observed on clinical examination of any of the ADPKD patients. DNA Electrophoresis and Hybridisation DNA extraction, restriction digests, electro¬ phoresis. Southern blotting, hybridisation and washing were performed by standard methods or as previously described (Harris, et al., 1990). FIGE was performed with the Biorad FIGE Mapper using programme 5 to -separate fragments from 25-50 kb . High molecular weight DNA for PFGE was isolated in agarose blocks and separated on the Biorad CHEF DRII apparatus using appropriate conditions . Genomic DNA probes and somatic cell hybrids Many of the DNA probes used in this study have been described previously: MS205.2 (D16S309; Royle, et al., 1992); GGGl (D16S259; Germino, et al. , 1990); N54 (D16S139; Himmelbauer, et al., 1991); SM6 (D16S665), CW23, CW21, and JHl (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). Microsatellite probes for haplotype analysis were KG8 and W5.2 (Snarey, et al., 1994) SM6, CW3 and CW2, (Peral, et al., 1994), 16AC2.5 (Thompson, et al. , 1992); SM7 (Harris, et al . , 1991), VK5AC (Aksentijevich, et al. , 1993). New probes isolated during this study were: JH4, JH5, JH6, 11 kb, 6 kb and 6 kb BamH I fragments, respectively, and JH13 and JH14, 4 kb and 2.8 kb BamH I-EcoR I fragments, respectively, all from the cosmid JH2A; JH8 and JH10 are 4.5 kb and 2 kb Sac I fragments, respectively and JH12 a 0.6 Sac I-BamH I fragment, all from JH4; 8S1 and 8S3 are 2.4 kb and 0.6 kb Sac II fragments, respectively, from JH8; CW10 is a 0.5 kb Not I-Mlu I fragment of SM25A; JH17 is a 2 kb EcoR I fragment of NM17. The somatic cell hybrids N-OH1 (Germino, et al., 1990) , P-MWH2A (European Chromosome 16 Tuberous Sclerosis Consortium, 1993) and Hyl45.19 (Himmelbauer, et al., 1991) have previously been described. Somatic cell hybrids containing the paternally derived (BP2-10) and maternally derived (BP2-9) chromosomes from 0X114 were produced by the method of Deisseroth and Hendrick (1979) . Constructing a cosmid contig Cosmids were isolated from chromosome 16 specific and total genomic libraries, and a contig was constructed using the methods and libraries previously described (European Chromosome 16 Tuberous Sclerosis Consortium, 1993). To ensure that cosmids were derived from the 16pl3.3 region (not the duplicate 16pl3.1 area) initially, probes from the single copy area were used to screen libraries (e.g. CW21 and N54) . Two cosmids mapped entirely within the area duplicated, CWIOIII and JC10.2B. To establish that these were from the PKDl area, they were restriction mapped and hybridised with the probe CWIO. The fragment sizes detected were compared to results obtained with hybrids containing only the 16pl3.3 area (Hyl45.19) or only the 16pl3.1 region (P-MWH2A) . FISH FISH was performed essentially as previously described (Buckle and Rack, 1993). The hybridisation mixture contained 100 ng of biotin-II-dUTP labelled cosmid DNA and 2.5 mg human Cot-1 DNA (BRL), which was denatured and annealled at 37°C for 15 min prior to hybridisation at 42°C overnight. After stringent washes the site of hybridisation was detected with successive layers of fluorescein-conjugated avidin (5 mg/ml) and biotinylated anti-avidin (5 mg/ml) (Vector Laboratories) . Slides were mounted in Vectashield (Vector Laboratories) containing 1 mg/ml propidium iodide and 1 mg/ml 4 ' , 6-diamidino-2-phenylindole (DAPI), to allow concurrent G-banded analysis under UV light. Results were analysed and images captured using a Bio-Rad MRC 600 confocal laser scanning microscope. cDNA screening and characterisation Foetal brain cDNAs libraries in 1 phage (Clonetech and Stratagene) were screened by standard methods with genomic fragments in the single copy area (equivalent to CW23 and CW21) or with a 0.8 kb Pvu II-Eco RI single copy fragment of AH3. Six PBP cDNAs were characterised including two previously described, AH4 (1.7 kb) , 3A3 (2.0 kb) (European Chromosome 16 Tuberous Sclerosis Consortium, 1993), and four novel cDNAs AH3 (2.2 kb), AH6 (2.0 kb), AIC (2.2 kb ) and B1E (2.9 kb) . A Striatum library (Stratagene) was screened with JH4 and a HG-C cDNA , 11BHS21 (3.8 kb) was isolated; 2IP.9 is a 0.9 kb Pvu II-EcoR I subclone of this cDNA. A HG-A or HG-B cDNA, HG-4 (7 kb ) was also isolated by screening the foetal brain library (Stratagene) with JH8. HG-4/1.1 is a 1.1 kb Pvu II-EcoR I fragment from the 3' end of HG-4. 1A1H.6 is a 0.6 kb Hind III-EcoR I subclone of a TSC2 cDNA, 1A-1 (1.7 kb) , which was isolated from the Clonetech library. Each cDNA was subcloned into Bluescript and sequenced utilising a combination of sequential truncation and oligonucleotide primers using DyeDeoxy Terminators (Applied Biosystems) and an ABI 373A DNA Sequencer (Applied Biosystems) or by hand with 'Sequenase' T7 DNA polymerase (USB) . RNA Procedures Total RNA was isolated from cell lines and tissues by the method of Chomczynski and Sacchi (1987) and enrichment for mRNA made using the PolyAT tract mRNA Isolation System (Promega). For RNA electrophoresis 0.5% agarose denaturing formaldehyde gels were used which were Northern blotted, hybridised and washed by standard procedures. The 0.24 - 9.5 kb RNA (Gibco BRL) size standard was used and hybridisation of the probe (1-9B3) to the 13 kb Utrophin transcript (Love, et al., 1989) in total fibroblast RNA was used as a size marker for the large transcripts. RT-PCR was performed with 2.5 mg of total RNA by the method of Brown et al (1990) with random hexamer primers, except that AMV-reverse transcriptase (Life Sciences) was employed. To characterise the deletion of the PBP transcript in 0X114 we used the primers : - 40 - AH3 F9 5 ' TTT GAC AAG CAC ATC TGG CTC TC 3 ' AH3 B7 5 ' TAC ACC AGG AGG CTC CGC AG 3 ' in a DMSO containing PCR buffer (Dode, et al . , 1990) with 0.5 mM MgCl 2 and 36 cycles of: 94°C, 1 min; 61°C, 1 min; 72°C, 2 min plus a final extension of 10 min. The 3A3 C primers used to amplify the 0X32 cDNA and DNA were: 3A3 CI 5' CGC CGC TTC ACT AGC TTC GAC 3' 3A3 C2 5' ACG CTC CAG AGG GAG TCC AC 3' These were employed in a PCR buffer and cycle previously described (Harris, et al . , 1991) with lmM MgCl 2 and an annealing temperature of 61°C. PCR products for sequencing were amplified with Pfu-1 (Stratagene) and ligated into the Srf-1 site in PCR-Script (Stratagene) in the presence of Srf-1. RNAse protection Tissues from normal and end-stage polycystic kidneys were immediately homogenised in guanidinium thiocyanate. RNA was purified on a cesium chloride gradient and 30 mg total RNA was assayed by RNAse protection by the method of Melton, et al. , (1984) using a genomic template generated with the 3A3 , C primers . Heteroduplex Analysis Heteroduplex analysis was performed essentially as described by Keen et al (1991). Samples were amplified from genomic DNA with the 3A3 , C primers, heated at 95°C for 5 minutes and incubated at room temperature for at least 30 minutes before loading on a Hydrolink gel (AT Biochem) . Hydrolink gels were run for 12-18 hours at 250V and fragments observed after staining with ethidium bromide. Extraction and amplification of paraffin-embedded DNA DNA from formalin f xed, paraffin wax embedded kidney tissue was prepared oy the method of Wright and Manos (1990), except that after proteinase K digestion overnight at 55°C, the DNA was extracted with phenol plus chloroform before ethanol precipitation. Approximately 50 ng of DNA was used for PCR with 1.5 mM MgCl and 40 cycles of 94°C for 1 min, 59°C for 1 min and 72°C for 40 s, plus a 10 min extension at 72°C. The oligonucleotide primers designed to amplify across the genomic deletion of 0X875 were: AH4F2 : 5' - GGG CAA GGG AGG ATG ACA AG - 3 ' JH14B3 : 5' - GGG TTT ATC AGC AGC AAG CGG - 3' which produced a product of ~ 220 bp in individuals with the 0X875 deletion. 3*RACE analysis of WS-212 3 ' RACE was completed essentially as described (European Polycystic Kidney Disease Consortium (1994)). Reverse transcription was performed with 5μg total RNA with 0.5μg of the hybrid dT^ 7 adapter primer using conditions previously described (Fronman et al . , (1988)). A specific 3' RACE product was amplified with the primer F5 adn adapter primer in 0.5mM MgCl 2 with the program: 57°C, 60s; 72°C, 15 minutes and 30 cycles of 95°C, 40s; 57°C, 60s; 72°C, 60s plus 72°C, 10 minutes. The amplified product was cloned using the TA cloning system (Invitrogen) and sequenced by conventional methods. References Aksenti jevich, I., Pras , E., Gruberg, L., Shen, Y., Holman, K., Helling, S., Prosen, L., Sutherland, G.R., Richards, R.I., Ramsburg, M. , Dean, M., Pras, M. , Amos, C.I. and Kastner, D.L. (1993). Refined mapping of the gene causing familial Mediterranean fever, by linkage and homozygosity studies. Am. J. Hum. Genet. 53, 451-461. Altschul, S.F., Warren, G., Miller, W. , Myers, E.W. and Lipman, D.J. (1990). Basic alignment search tool. J. Mol. Biol. 215, 403-410. Breuning, M.H., Reeders, S.T., Brunner, H., Ijdo, J.W., Saris, J.J., Verwest, A., van Ommen, G.J.B. and Pearson, P.L. (1987). Improved early diagnosis of adult polycystic kidney disease with flanking DNA markers. Lancet, ii, 1359-1361. 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Claims: CLAIMS 1. An isolated, purified or recombinant nucleic acid sequence comprising:- (a) a PKDl gene or its complementary strand, (b) a sequence substantially homologous to, or capable of hybridising to, a substantial portion of a molecule defined in (a) above, (c) a fragment of a molecule defined in (a) or (b) above. 2. A sequence according to claim 1, wherein the PKDl gene has the partial nucleic acid sequence according to Figure 7 and/or 10. 3. A sequence according to claim 1 or claim 2 comprising a DNA molecule selected from: (a) a PKDl gene or its complementary strand, (b) a sequence substantially homologous to, or capable of hybridising to, a substantial portion of a molecule defined in (a) above, (c) a molecule coding for a polypeptide having the partial sequence of Figure 7, (d) genomic DNA corresponding to a molecule in (a) above; and (e) a fragment of a molecule defined in any of (a), (b), (c) or (d) above. 4. A nucleic acid sequence comprising a mutant PKD l gene, selected from those wherein:- (a) [0X114] base pairs 1746-2192 as defined in Figure 7 are deleted (446bp); (b) [0X32] base pairs 3696-3831 as defined in Figure 7 are deleted by a splicing defect; (c) [0X875] about 5.5kb flanked by the two Xbal sites shown in Figure 3a are deleted and the EcoRl site separating the CWIO (41kb) and JHl (18kb) sites is thereby absent; and (d) [WS53] about lOOkb extending between the JHl and CW21 and the S 6 and JHl7 sites shown in Figure 6 and the PKDl gene is thereby absent. 5. A nucleic acid sequence comprising a mutant PKDl gene selected from those wherein- (a) [461] abpout 18bp are deleted in the 75bp intron amplified by the primer pair 3A3C insert at position 3696 of the 3' sequence as shown in Figure 11; (b) [0X1054] about 20bp are deleted in the 75bp intron amplified by the primer pair 3A3C insert at position 3696 of the 3' sequence as shown in Figure 11; (c) [WS212] about 75kb are deleted between SM9-CW9 distally and the PKDl 3'UTR proximally as shown in Figure 12; (d) [WS-215] about 160kb are deleted between CW20 and CW10-CW36 as shown in Figure 12; (e) [WS-227] about 50kb are deleted between CW20 and JH11 as shown in Figure 12; (f) [WS-219] about 27kb are deleted between JHl and JH6 as shown in Figure 12; and (g) [WS-250] about 160kb are deleted betwenn WC20 and B u24 as shown in Figure 12. (h) [WS194] a deletion of about 65kb between CW20 and CWIO. 6. An RNA molecule comprising an RNA sequence corresponding to a DNA sequence according to any of claims 1 to 5. 7. An RNA molecule according to claim 6, wherein the molecule is the transcript referenced PKDl and identifiable from the restriction map of Figure 3a and having a sequence of about 14 KB. 8. A nucleic acid probe having a sequence according to any of the preceding claims and optionally including a label. 9. A nucleic acid sequence according to any preceding claim, wherein the nucleic acid sequence encoding PKDl is operably linked to transcriptional and/or translational expression signals. 10. An isolated, purified or recombinant polypeptide comprising a PKDl protein or a mutant or variant thereof or encoded by a sequence according to any of claims 1 to 9 or a variant thereof having substantially the same activity as the PKDl protein. 11. A polypeptide according to claim 10, wherein the PKDl protein has the amino acid sequence according to the partial amino acid sequence of Figure 7 and/or Figure 10. 12. An anti-PKDl antibody or a labelled anti-PKDl antibody. 13. A method for screening a subject to determine whether said subject is a PKDl-associated disorder carrier or a patient having a PKDl-associated disorder, which method comprises detecting the presence of and/or evaluating the characteristics of PKDl DNA, PKDl RNA and/or PKDl polypeptide in a biological sample from said patient. 14. A method according to claim 13 which is or includes detecting and/or evaluating whether the PKDl DNA is mutated, deleted, aberrant or otherwise abnormal, or is not expressing normal PKDl protein. 15. A method according to claim 13 or claim 14, wherein the detection and/or evaluation includes the step of comparing the results thereof with results obtained using a mutated sequence according to claim 4 or claim 5. 16. A method according to any of claims 13 to 15, wherein said screening includes applying a nucleic acid amplification process to said sample to amplify a fragment of the PKDl DNA or cDNA corresponding to the PKDl RNA. 17. A method according to claim 16, wherein said nucleic acid amplification process uses at least one of the following sets of primers as identified herein:- AH3 F9 : AH3 B7 3A3 CI : 3A3 C2 AH4 F2 : JH14 B3 18. A method according to any of claims 13 to 17 which comprises digesting said sample to EcoRl fragments and hybridising with a DNA probe which hybridises to the EcoRl fragment identified (A) in Figure 3(a). 19. A method according to claim 18, wherein said DNA probe comprises the DNA probe CWIO identified herein. 20. A method according to any of claims 13 to 17 which comprises digesting said sample to provide BamHI fragments hybridising with a DNA probe which hybridises to the BamHI fragment identified (B) in Figure 3(a). 21. A method according to claim 20, wherein said DNA probe comprises the DNA probe 1A1H.6 identified herein. 22. A vector (such as Bluscript (available from Stratagene)) comprising the nucleic acid sequence of any of claims 1 to 9. 23. A host cell (such as E. coli strain SL-1 Blue (available from Stratgene)) transfected or transformed with a vector according to claim 22. 24. The use of a vector according to claim 23 or a nucleic acid sequence according to any of claims 1 to 11 in gene therapy and/or in the preparation of an agent for treating or preventing a PKDl-associated disorder. 25. A method of treating or preventing a PKDl- associated disorder which method comprises administering to a patient in need thereof a functional PKDl gene to affected cells in a manner that permits expression of PKDl protein therein and/or a transcript produced from a mutated chromosome such as the deleted WS-212 chromosome which is capable of expressing functional PKDl protein therein. 26. A diagnostic kit for carrying out a method according to any of claims 13 to 21, comprising nucleic acid primers for amplifying a fragment of a sequence according to any of Claims 1 to 9. 27. A diagnostic kit according to claim 26, wherein the nucleic acid primers comprise at least one of the following sets: AH3 F9 : AH3 B7 3A3 CI : 3A3 C2 AH4 F2 : JH14 B3 28. A diagnostic kit for carrying out a method according to claim 18, including one or more substances for digesting a sample to provide EcoRl fragments and a DNA probe as defined in claim 19. 29. A diagnostic kit for carrying out a method according to claim 20, including one or more substances for digesting a sample to provide BamHI fragments and a DNA probe as defined in claim 21. 30. A diagnostic kit for carrying out a method for determining whether said subject is a PKDl-associated disorder carrier or a patient having a PKDl-associated disorder, which includes a nucleic acid probe capable of hybridising to a sequence according to any of claims 1 to 11. SUBSTITUTE SHEET tøUiE 26)

Inventors: BREUNING MARTIN HENDRIK, HALLEY DIRKJE JORIJNTJE JOHANN, HARRIS PETER CHARLES, HESSELING AJENNE LIQUE WILHELM, HUGHES JAMES, JANSSEN LAMBERTUS ANTONIUS JAC, NELLIST MARK DAVID, PERAL BELEN, PETERS DOROTHEA JOHANNA MARIA, ROELFSEMA JEROEN HENDRIK, SAMPSON JULIAN, WARD CHRISTOPHER JAMES, BREUNING, MARTIN, HENDRIK, HALLEY, DIRKJE, JORIJNTJE, JOHANNA, HARRIS, PETER, CHARLES, HESSELING, AJENNE, LIQUE, WILHELMA, HUGHES, JAMES, JANSSEN, LAMBERTUS, ANTONIUS, JACOBUS, NELLIST, MARK, DAVID, PERAL, BELEN, PETERS, DOROTHEA, JOHANNA, MARIA, ROELFSEMA, JEROEN, HENDRIK, SAMPSON, JULIAN, WARD, CHRISTOPHER, JAMES
Applicants: BREUNING MARTIN HENDRIK, HALLEY DIRKJE JORIJNTJE JOHANN, HARRIS PETER CHARLES, HESSELING AJENNE LIQUE WILHELM, HUGHES JAMES, JANSSEN LAMBERTUS ANTONIUS JAC, MEDICAL RES COUNCIL, NELLIST MARK DAVID, PERAL BELEN, PETERS DOROTHEA JOHANNA MARIA, ROELFSEMA JEROEN HENDRIK, SAMPSON JULIAN, UNIV ERASMUS, UNIV LEIDEN, UNIV WALES MEDICINE, WARD CHRISTOPHER JAMES, BREUNING, MARTIN, HENDRIK, ERASMUS UNIVERSITY ROTTERDAM, HALLEY, DIRKJE, JORIJNTJE, JOHANNA, HARRIS, PETER, CHARLES, HESSELING, AJENNE, LIQUE, WILHELMA, HUGHES, JAMES, JANSSEN, LAMBERTUS, ANTONIUS, JACOBUS, LEIDEN UNIVERSITY, MEDICAL RESEARCH COUNCIL, NELLIST, MARK, DAVID, PERAL, BELEN, PETERS, DOROTHEA, JOHANNA, MARIA, ROELFSEMA, JEROEN, HENDRIK, SAMPSON, JULIAN, UNIVERSITY OF WALES COLLEGE OF MEDICINE, WARD, CHRISTOPHER, JAMES
Application year: 1994
Main classification: C12N15
IPC codes: A61K31, A61K38, A61K39, A61K48, A61P13, A61P15, A61P35, C07H21, C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12Q1, C12R1, G01N33